Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract leukocytes, as illustrated by macrophages, T cells, B cells, eosinophils, basophils, and neutrophils to and from sites of inflammation or within specific compartments, as illustrated by lymph nodes (reviewed in Schall, Cytokine 1991; 3:165-183; Schall, et al., Curr. Opin. Immunol. 1994; 6:865-873; and Murphy, Rev. Immun. 1994; 12:593-633). In addition to stimulating chemotaxis, other changes can be selectively induced by chemokines in responsive cells, including changes in cell shape, transient rises in the concentration of intracellular free calcium ions ([Ca2+]), granule exocytosis, integrin upregulation, formation of bioactive lipids (e.g., leukotrienes), and respiratory burst, associated with leukocyte activation. Thus, the chemokines are early modulators of inflammatory response, effecting inflammatory mediator release, chemotaxis and extravasation to sites of infection or inflammation.
There are four classes of chemokines, CXC (α), CC (β), C (γ), and CX3C (δ), depending on whether the first two cysteines are separated by a single amino acid (C—X—C), are adjacent (C—C), have a missing cysteine pair (C), or are separated by three amino acids (CX3C). The α-chemokines, such as interleukin-8 (IL-8), melanoma growth stimulatory activity protein (MGSA), and stromal cell derived factor 1 (SDF-1) are chemotactic primarily for neutrophils and lymphocytes, whereas 1-chemokines, such as RANTES, MIP-1α, MIP-10, monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, and eotaxin are chemotactic for macrophages, T-cells, eosinophils and basophils (Deng, et al., Nature 1996; 381:661-666). The C chemokine lymphotactin shows specificity for lymphocytes (Kelner, et al., Science 1994; 266:1395-1399) while the CX3C chemokine fractalkine shows specificity for lymphocytes and monocytes (Bazan, et al., Nature 1997; 385:640-644).
Chemokines bind specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins (reviewed in Horuk, Trends Pharm. Sci. 1994; 15:159-165) termed “chemokine receptors.” On binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated heterotrimeric G protein, resulting in a rapid increase in intracellular calcium concentration. There are at least twelve human chemokine receptors that bind or respond to β-chemokines with the following characteristic pattern: CCR1 (or “CKR-1” or “CC-CKR-1”) MIP-1α, MIP-13, MCP-3, RANTES (Ben-Barruch, et al., J. Biol. Chem. 1995; 270:22123-22128; Neote, et al., Cell 1993; 72:415425); CCR2A and CCR2B (or “CKR-2A”/“CKR-2A” or “CC-CKR-2A”/“CC-CKR2A”) MCP-1, MCP-2, MCP-3, MCP-4; CCR3 (or “CKR-3” or “CC-CKR-3”) eotaxin, RANTES, MCP; (Ponath, et al., J. Exp. Med. 1996; 183:2437-2448); CCR4 (or “CKR-4” or “CC-CKR-4”) TARC, MDC (Imai, et al., J. Biol. Chem. 1998; 273:1764-1768); CCR5 (or “CKR-5” or “CC-CKR-5”) MIP-1α, RANTES, MIP-1β; (Sanson, et al., Biochemistry 1996; 35:3362-3367); CCR6MIP-3α (Greaves, et al., J. Exp. Med. 1997; 186:837-844); CCR7 MIP-3β and 6Ckine (Campbell, et al., J. Cell. Biol. 1998; 141:1053-1059); CCR8 I-309, HHV8 vMIP-I, HHV-8 vMIP-II, MCV vMCC-I (Dairaghi, et al., J. Biol. Chem. 1999; 274:21569-21574); CCR9TECK (Zaballos, et al., J. Immunol. 1999; 162:5671-5675), D6 MIP-1 beta, RANTES, and MCP-3 (Nibbs, et al., J. Biol. Chem. 1997; 272:32078-32083), and the Duffy blood-group antigen RANTES, MCP-1 (Chaudhun, et al., J. Biol. Chem. 1994; 269:7835-7838).
Chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, and XCR1 have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.
The CCR2 chemokine receptor is expressed primarily in monocytes and activated T lymphocytes, and its functional activity can be measured by cytosolic calcium elevation or chemotaxis. CCR2 exists in two isoforms, CCR2A and CCR2B. These two isoforms are alternatively spliced variants of a single MCP-1 receptor gene and differ only in the carboxyl-terminal tails. The chromosomal location of the CCR2 gene is localized to 3p21. The CC chemokines, MCP-1, MCP-2, MCP-3, and MCP-4, have been identified as the ligands that are selective and of high affinity to the CCR2 receptor.
The highly selective expression of CCR2 makes it an ideal target for intervention to interrupt inappropriate monocyte and T cell trafficking. The clinical indications for such intervention are in inflammatory diseases and T-cell mediated autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, asthma, allergy, chronic obstructive pulmonary disease, atherosclerosis, restinosis, type I and type II diabetes, metabolic syndrome, and pain. Ectopic expression of MCP-1 and CCR2 in certain tumors indicates that selective modulation (such as antagonism or inhibition) of CCR2 can have value in tumor immunotherapy, particularly attenuation of metastasis.
The native peptide ligand of CCR2 is monocyte chemoattractant protein-1 (MCP-1 or CCL2) containing two adjacent disulfide bonds. Ample evidence exists for the role of the CCR2/MCP-1 system in preclinical animal models of pain (White F. A., Jung F., and Miller R. J., Proc. Natl. Acad. Sci. USA 2007; 51:20151). Although CCR2 and MCP-1 have limited expression levels in the CNS tissues under normal conditions, significant upregulation of CCR2 and MCP-1 has been observed following a neuropathic injury in tissue relevant to pain, including neurons and glia in the spinal cord, rostroventromedial medulla (RVM) and DRG (Wang H., Zou S., Wei F., Dubner R., and Ren K., Soc for Neurosci Poster 2009; 72.3). MCP-1 has been shown to increase the excitability of neurons acutely dissociated from the DRG tissue (Sun J. H., Yang B., Donnelly D. F., Ma C., and LaMotte R. H., J Neurophysiol. 2006; 96:2189). In addition, direct injection of MCP-1 in the spinal cord induces thermal hyperalgesia and mechanical allodynia (Dansereau et al. Neurochem. 2008; 106:7), and the MCP-1 induced pronociception can be blocked by a CCR2 antagonist, INCB3344. Similarly, the hyperalgesia induced by MCP-1 injection in the RVM is reversed by another CCR2 antagonist, RS102895 (Wang H., Zou S., Wei F., Dubner R. and Ren K., Soc for Neurosci Poster 2009; 72.3). In addition, CCR2 knock out mice exhibit significantly reduced mechanical allydonia following nerve injury and reduced nocifensive behavior in the second phase of the formalin model, whereas they exhibit normal sensitivity to acute pain stimulation in the hot plate model (Abbadie C., Lindia J. A., Cumiskey A. M., Peterson L. B., Mudgett J. S., Bayne E. K., DeMartino J. A., Maclntyre D. E., and Forrest M. J., Proc Natl Aca Sci USA 2003; 100:7947). Treatment with AZ889 (Serrano A., Pare M., McIntosh F., Elmes S. J. R. Martino G., Jomphe C., Lessard E., Lembo P. M. C., Vaillancourt F., Perkins M. N., and Cao C. Q., Mol. Pain. 2010; 6:90), a CCR2 antagonist, abolished CCL2-evoked neuronal excitation, confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. In vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in chronic constriction injury rats (Serrano A., Paré M., McIntosh F., Elmes S. J. R., Martino G., Jomphe C., Lessard E., Lembo P. M. C., Vaillancourt F., Perkins M. N., and Cao C. Q., Mol. Pain. 2010; 6:90). Additionally, application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord.
In view of the clinical importance of CCR2, the identification of compounds that modulate CCR2 function represents an attractive avenue into the development of new therapeutic agents that can be used to treat diseases that are associated with chemokine receptor expression or activity such as inflammatory, autoimmune disease, cancer, and pain. Such compounds are provided herein.